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The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin.

Qin Y, Capaldo C, Gumbiner BM, Macara IG - J. Cell Biol. (2005)

Bottom Line: These effects are independent of Rac activation or Scrib binding to betaPIX.Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion.Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to betaPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

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Loss of Scrib causes a defect in adherens junction structure. (A) Control and Scrib-depleted cells were plated at subconfluence and allowed to form islands of cells. They were then fixed and stained for E-cadherin (red) and Na/K-ATPase (green). Image stacks were collected using confocal microscopy with 0.95-μm z steps. (B) Control and ScrbKD cells were fixed and stained for Scrib and β-catenin. (C) Immunoblot of junctional proteins in control and ScrbKD cells. Whole cell lysates were blotted for E-cadherin, α- and β-catenin, and α-tubulin. (D) Cells were treated with a nonpermeable biotin linker, sulfo-NHS-SS-biotin, and biotinylated proteins were captured onto streptavidin-agarose and detected by immunoblot. (E) Control and ScrbKD cells plated at low density were lysed with buffer containing 0.5% Triton X-100. After centrifugation, supernatants and pellets were resolved by SDS-PAGE. Distributions of α- and β-catenin were detected with immunoblot. 25% of the soluble and 50% of the insoluble fractions are shown. (F) Quantification by Odyssey image system. Data are presented as the ratio of insoluble to soluble fractions. Error bars represent the SD from four independent experiments.
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fig7: Loss of Scrib causes a defect in adherens junction structure. (A) Control and Scrib-depleted cells were plated at subconfluence and allowed to form islands of cells. They were then fixed and stained for E-cadherin (red) and Na/K-ATPase (green). Image stacks were collected using confocal microscopy with 0.95-μm z steps. (B) Control and ScrbKD cells were fixed and stained for Scrib and β-catenin. (C) Immunoblot of junctional proteins in control and ScrbKD cells. Whole cell lysates were blotted for E-cadherin, α- and β-catenin, and α-tubulin. (D) Cells were treated with a nonpermeable biotin linker, sulfo-NHS-SS-biotin, and biotinylated proteins were captured onto streptavidin-agarose and detected by immunoblot. (E) Control and ScrbKD cells plated at low density were lysed with buffer containing 0.5% Triton X-100. After centrifugation, supernatants and pellets were resolved by SDS-PAGE. Distributions of α- and β-catenin were detected with immunoblot. 25% of the soluble and 50% of the insoluble fractions are shown. (F) Quantification by Odyssey image system. Data are presented as the ratio of insoluble to soluble fractions. Error bars represent the SD from four independent experiments.

Mentions: These data suggest that Scrib is required for normal E-cadherin function at cell–cell junctions. We therefore examined the distribution of E-cadherin and of the Na/K-ATPase, which is a marker for the basolateral membrane. In control cells, these proteins colocalize along the lateral cell boundaries. Scrib KD caused a distinctive phenotype in which the lateral membranes of the cells became disorganized. The membranes appeared less vertical and had convoluted edges (Fig. 7 A). A similar phenotype was observed for β-catenin distribution (Fig. 7 B). However, the total amounts of E-cadherin, β-catenin, and α-catenin expressed in cells depleted of Scrib were the same as the amounts in control cells (Fig. 7 C). Scrib does not, therefore, regulate the expression of these junctional proteins. Moreover, when surface proteins were biotinylated, captured on streptavidin beads, and blotted for E-cadherin, no reproducible difference was observed between the control and Scrib KD cells (Fig. 7 D). These data demonstrate that there is no change in the amount of E-cadherin on the cell surface, and we conclude that Scrib is not involved in controlling the exocytosis or endocytosis of E-cadherin.


The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin.

Qin Y, Capaldo C, Gumbiner BM, Macara IG - J. Cell Biol. (2005)

Loss of Scrib causes a defect in adherens junction structure. (A) Control and Scrib-depleted cells were plated at subconfluence and allowed to form islands of cells. They were then fixed and stained for E-cadherin (red) and Na/K-ATPase (green). Image stacks were collected using confocal microscopy with 0.95-μm z steps. (B) Control and ScrbKD cells were fixed and stained for Scrib and β-catenin. (C) Immunoblot of junctional proteins in control and ScrbKD cells. Whole cell lysates were blotted for E-cadherin, α- and β-catenin, and α-tubulin. (D) Cells were treated with a nonpermeable biotin linker, sulfo-NHS-SS-biotin, and biotinylated proteins were captured onto streptavidin-agarose and detected by immunoblot. (E) Control and ScrbKD cells plated at low density were lysed with buffer containing 0.5% Triton X-100. After centrifugation, supernatants and pellets were resolved by SDS-PAGE. Distributions of α- and β-catenin were detected with immunoblot. 25% of the soluble and 50% of the insoluble fractions are shown. (F) Quantification by Odyssey image system. Data are presented as the ratio of insoluble to soluble fractions. Error bars represent the SD from four independent experiments.
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Related In: Results  -  Collection

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fig7: Loss of Scrib causes a defect in adherens junction structure. (A) Control and Scrib-depleted cells were plated at subconfluence and allowed to form islands of cells. They were then fixed and stained for E-cadherin (red) and Na/K-ATPase (green). Image stacks were collected using confocal microscopy with 0.95-μm z steps. (B) Control and ScrbKD cells were fixed and stained for Scrib and β-catenin. (C) Immunoblot of junctional proteins in control and ScrbKD cells. Whole cell lysates were blotted for E-cadherin, α- and β-catenin, and α-tubulin. (D) Cells were treated with a nonpermeable biotin linker, sulfo-NHS-SS-biotin, and biotinylated proteins were captured onto streptavidin-agarose and detected by immunoblot. (E) Control and ScrbKD cells plated at low density were lysed with buffer containing 0.5% Triton X-100. After centrifugation, supernatants and pellets were resolved by SDS-PAGE. Distributions of α- and β-catenin were detected with immunoblot. 25% of the soluble and 50% of the insoluble fractions are shown. (F) Quantification by Odyssey image system. Data are presented as the ratio of insoluble to soluble fractions. Error bars represent the SD from four independent experiments.
Mentions: These data suggest that Scrib is required for normal E-cadherin function at cell–cell junctions. We therefore examined the distribution of E-cadherin and of the Na/K-ATPase, which is a marker for the basolateral membrane. In control cells, these proteins colocalize along the lateral cell boundaries. Scrib KD caused a distinctive phenotype in which the lateral membranes of the cells became disorganized. The membranes appeared less vertical and had convoluted edges (Fig. 7 A). A similar phenotype was observed for β-catenin distribution (Fig. 7 B). However, the total amounts of E-cadherin, β-catenin, and α-catenin expressed in cells depleted of Scrib were the same as the amounts in control cells (Fig. 7 C). Scrib does not, therefore, regulate the expression of these junctional proteins. Moreover, when surface proteins were biotinylated, captured on streptavidin beads, and blotted for E-cadherin, no reproducible difference was observed between the control and Scrib KD cells (Fig. 7 D). These data demonstrate that there is no change in the amount of E-cadherin on the cell surface, and we conclude that Scrib is not involved in controlling the exocytosis or endocytosis of E-cadherin.

Bottom Line: These effects are independent of Rac activation or Scrib binding to betaPIX.Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion.Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to betaPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

Show MeSH
Related in: MedlinePlus